Modulating neuroinflammation through molecular, cellular and biomaterial‐based approaches to treat spinal cord injury

Abstract The neuroinflammatory response that is elicited after spinal cord injury contributes to both tissue damage and reparative processes. The complex and dynamic cellular and molecular changes within the spinal cord microenvironment result in a functional imbalance of immune cells and their modulatory factors. To facilitate wound healing and repair, it is necessary to manipulate the immunological pathways during neuroinflammation to achieve successful therapeutic interventions. In this review, recent advancements and fresh perspectives on the consequences of neuroinflammation after SCI and modulation of the inflammatory responses through the use of molecular‐, cellular‐, and biomaterial‐based therapies to promote tissue regeneration and functional recovery will be discussed.

dynamic pathophysiological events after SCI, especially a cascade of immunological responses resulting from neuroinflammation, pose as a major challenge for many therapeutic interventions, including celland biomaterial-based therapies. 11 Thus, a comprehensive review of the recent advancements in the cellular and molecular mechanisms involved in neuroinflammation after SCI is crucial to develop strategic interventions against this debilitating condition.

| NEUROINFLAMMATION AFTER SCI
Neuroinflammation is defined as an inflammatory response that occurs within the brain or spinal cord. Upon damage to the bloodspinal cord barrier (BSCB) after a physical trauma, neuroinflammation is one of the key components during the primary phase, which persists towards the secondary phase of injury. 8,12 The acute period of neuroinflammation is characterized by an infiltration of neutrophils and monocytes to the site of injury, 13 whereas in the chronic phase, the progressive tissue degeneration that takes place across a period of months is primarily driven by lymphocytes. 14 Inflammatory responses play a central role in regulating the pathophysiology after SCI, which greatly contributes to the repair of damaged tissues. 15,16 However, excessive inflammation may also lead to apoptosis of neurons and oligodendrocytes, resulting in a decline in neuronal functions. 16 Inevitably, changes within the spinal cord microenvironment during neuroinflammation may aggravate and accelerate the course of SCI.

| MICROENVIRONMENT CHANGES DURING NEUROINFLAMMATION
During neuroinflammation, a cascade of cellular and molecular inflammatory pathways is activated, which includes the influx of circulating immune cells (neutrophils, monocytes, and lymphocytes), activation and proliferation of resident microglia and astrocytes, and the production of several mediators such as cytokines, chemokines, and reactive oxygen species by immune cells that reside in the central nervous system (CNS; Figure 1). 8,12,14,17 Paradoxically, while these secreted molecules are important in re-establishing tissue homeostasis and assisting in wound healing and repair, 18,19 there are also collateral effects of secondary damage by inhibiting axonal regeneration or causing neuronal hypersensitivity, leading to neuropathic pain. [20][21][22] Together, this imbalance may impair regenerative capacity and functional recovery.

| Peripheral immune cells
Within a few hours after SCI, the first immune cell type to arrive at the site of injury is the neutrophils. 12 They secrete oxidative and proteolytic enzymes to sterilize the lesion and prepare the tissue for subsequent repair. 12 However, their presence is short-lived approximately 3-5 days, plausibly due to their neurotoxic nature as neutrophils release potent free radicals. 8,12 A few days after neutrophils infiltration, monocyte-derived macrophages are recruited to phagocytose dead cells including apoptotic neutrophils from the lesion. 23 Interestingly, these macrophages have been reported to also secrete factors such as resolvins and protectins to prevent further recruitment of neutrophils to the damaged tissue. 24 Unlike neutrophils, macrophages reside in the SCI lesions for as long as a year in humans. 12,25 While the recruitment of these innate immune cells serves to promote neuronal regeneration, wound healing, and tissue repair, both cell types instinctively produce proteases including matrix metalloproteinases (MMP), and oxidative metabolites that would compromise the BSCB. 12,26,27 On the other hand, adaptive immune cells such as the T-and Blymphocytes also infiltrate the lesion site, albeit only after weeks to months later. 12,14 SCI-induced T-lymphocytes typically have a life span of 1-2 months and are also involved in the recovery and regeneration of the spinal cord tissues. 28,29 Reportedly, T-lymphocytes elicit their neuroprotective capability through the recognition of specific neural antigens, such as myelin basic protein (MBP), whereby a drastic improvement in the rate of neuronal survival was observed. 30,31 However, even though T-lymphocytes are relatively lower in numbers than macrophages, they are also capable in inflicting tissue damage, albeit controversially, through the recognition of the same MBP antigen. 32 These opposing outcomes arise, depending on the spinal cord microenvironment at the time of injury, which would drive the equilibrium towards either a pathogenic Th1 or immunoregulatory Th2 lymphocytes expansion. 33 For instance, in the event of more regulatory T-lymphocytes recruitment to the lesion, there could be a more robust expression of neurotrophins, which would ameliorate the tissue damage induced by the secreted pro-inflammatory cytokines. 34 In association to an increase in T-lymphocytes infiltration, there is an acute upregulation of cell death-related genes and potassium voltage-gated channel-related (K v ) genes. 35,36 The high expression of K v genes such as contactin-2 (CNTN2) typically occurs in response to early demyelination in rats. 36 Furthermore, chronic T-cell activation is shown to be involved in pathological tissue fibrosis and scarring. 37 Since neural gene-specific proteins such as anti-MBP antibodies are detected after SCI, B-lymphocytes are also involved during neuroinflammation. 32 Mice deficient in B-lymphocytes exhibited an improved locomotor function and reduced spinal pathology, indicating a pathogenic role of these cells in spinal cord tissue repair. 38 The antibodies produced by SCI-induced B-lymphocytes are shown to be neurotoxic as the passive transfer of sera from SCI animals induced glial reactivity that is accompanied by prominent neuron loss. 14 Interestingly, concomitant tissue injury may induce anti-CNS antibodies that are able to promote axonal regeneration and remyelination. 14,39 For instance, antibodies targeting myelin may cause spinal cord demyelination, however, some antibodies prevent the binding by other myelin proteins that are inhibitory to axon growth and remyelination. 14,40 Together, there is a significant and long-term contribution of peripheral immune cells during neuroinflammation within the spinal cord microenvironment.

| Resident immune cells of the CNS
Apart from the peripheral circulating innate and adaptive immune cells, resident cells of the CNS, such as microglia and astrocytes, also play crucial roles during neuroinflammation after SCI. Having the same progenitor as tissue macrophages, the microglia comprise 10% of the population in the CNS. 42 These cells perform primary immunosurveillance functions of the tissue microenvironment, where they become elevated on the first day after SCI, and rapidly induce the production of cytokines and chemokines to recruit peripheral macrophages to the site of injury. 43-45 Trophic factors secreted by microglia are necessary for the survival and proliferation of infiltrating cells, as well as the growth and regeneration of axons in the spinal cord lesion. 46,47 At the same time, microglia may also help to prevent further expansion of the lesion site. 48 While the microglia responding to the damage after SCI is associated with tissue reorganization, it was reported to impede functional recovery of the neural tissue through the production of MMP-9, which has been widely reported to amplify pro-inflammatory cytokine secretion and affect the BSCB integrity, thereby interfering with plasticity and recovery. 49,50 F I G U R E 1 Schematic of the spinal cord microenvironment after spinal cord injury (SCI). (a) Within the first few hours after injury, inflammation occurs when peripheral immune cells begin infiltrating the lesion site, and resident immune cells become activated. Progressively, peri-lesion perimeters with multicellular components including astrocytes, neurons, macrophages, microglia, oligodendrocyte progenitor cells, fibroblast, and activated astrocytes start to form a compact astrocyte core, regulating the formation of a glial scar to restrict inflammation and protect the surrounding of the injured tissue. These scar-forming astrocytes serve as bridges for axonal regrowth, and structural tissue regeneration occurs weeks to months after SCI. (b) Timeline of both biological and molecular events following SCI. Illustrations are adapted from Donnelly and Popovich 41 and created with BioRender.com.
Astrocytes are found in two areas of SCI lesion: (1) tissues that are spared by injury and (2) scar borders. The phenotype and functions of the astrocytes are distinct in both compartments. 51 Astrocytes that reside in spared tissues are reactive, non-proliferative, and hypertrophic, and they primarily intermingle with neurons and synapses. 51 These hypertrophic astrocytes interact closely with neurons to promote axon sprouting and synapse plasticity through regulating the expression of neurocan, tenascin-C, or directly producing thrombospondin-1. [52][53][54] On the other hand, scar-forming astrocytes are majority spontaneously proliferated upon damage, where they interweave to create glia limitans borders that restrict inflammation and keep non-neural lesion core apart from adjacent functioning spinal cord tissue. 55,56 Surprisingly, axonal regeneration is not impeded by the presence of astrocyte scar formation as these scar-forming astrocytes may serve as bridges for axonal growth. 57 Instead, the disruption of the scar tissues, shown through the use of loss-of-function transgenic mice that selectively kill proliferating scar-forming astrocytes, led to an attenuation of axon growth after SCI. 58 Astrocyte scar borders are intertwined with reactive oligodendrocyte progenitor cells that express neuron glial antigen 2 (NG2-OPCs). Similarly, NG2-OPCs are also present in both the spared tissues and scar borders.
However, there have been several conflicting studies on axonal regrowth by these hypertrophic NG2-OPCs within the scar borders, 59-63 which warrant further investigations to understand the roles of these cells during neuroinflammation. Overall, the roles of these spinal cord neural cells play an important role in regulating tissue damage after SCI.

| Cytokines and chemokines
Cytokines are regulatory mediators that contribute immensely during neuroinflammation, neurodegeneration, and neuropathic pain through intricate cross-talks and interplays. 64 They are usually classified into proinflammatory or anti-inflammatory proteins, 64 although some cytokines may exhibit pro-inflammatory and anti-inflammatory properties under various circumstances. 65 Endogenous cells in the spinal cord, mainly the neurons, microglia, and astrocytes, support the early production of key inflammatory mediators, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNFα). [66][67][68][69] These pro-inflammatory cytokines, along with others including granulocyte-macrophage colony-stimulating factor (GM-CSF) and leukocyte inhibitory factor (LIF), contribute to the dynamic imbalance within the spinal cord microenvironment. 66,67,70 At low concentrations, these cytokines elicit protective functions by inducing neurotrophic factors and adhesion molecules on the cell surface, which assist in leukocyte recruitment to the injury site. 71 However, at a higher concentration, their pro-inflammatory nature typically causes neuronal damage and destruction through the activation of transcription factors that stimulate the expression of neurotoxic genes such as cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). 72,73 High amounts of IL-1 within the spinal cord microenvironment result in increased vascular permeability and lymphocyte recruitment, while IL-6 promotes the activation and infiltration of peripheral immune cells and microglia. 74 Blockade of IL-6 signaling was reported to enhance SCI recovery as it abrogates damaging inflammatory activity and reduces the severity of connective tissue scar formation. 74,75 TNFα is involved in several aspects of SCI neuroinflammation. Upon secretion, TNFα promotes the extravasation of neutrophils to the damaged tissue through adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1). 76 TNFα also induces changes to the permeability of endothelial cells, thereby compromising the integrity of the BSCB. 76 In addition, this pro-inflammatory cytokine exerts cytotoxic effects on oligodendrocytes, resulting in demyelination. 76 Furthermore, TNFα also contributes to fibrotic scarring by stimulating the proliferation and hypertrophy of astrocytes. 77 Anti-inflammatory cytokines including IL-4 and IL-10 are also produced to regulate and aid in functional recovery after SCI. 78 IL-4 is secreted by activated T-lymphocytes and is involved in the Th2 immunoregulatory pathway where it regulates the activation of acute macrophages and restrict secondary cavity formation after SCI. 79 In addition, IL-4 also drives microglia and macrophages toward an anti-inflammatory phenotype that reduces tissue damage, thereby leading to an improved functional recovery. 80 The production of IL-10 by monocytes/macrophages, astrocytes, and microglia functions to suppress the inflammatory responses through the reduction of TNFα, IL-1β, S100β, and iNOS. 15, 78,81 IL-10 is involved in regulating the influx and efflux of macrophages out of the injured nerve, reducing the production of pro-inflammatory chemokines and cytokines, and it is necessary for myelin-phagocytosis-induced shift of macrophages from pro-inflammatory to anti-inflammatory. 82 Furthermore, the loss of IL-10 affects axon regeneration, resulting in a poor recovery of motor and sensory functions. 82 More recently, a scaffold that comprise photocrosslinked gelatin hydrogel that was incorporated with polyamidoamine and IL-10 enhanced tissue remodeling and promoted axonal regeneration. 83 On the other hand, chemokines are small, secreted molecules that stimulate specific functions during inflammation. The kinetics of chemokine production usually parallel the infiltration of immune cells after SCI. 45 Chemokines that belong to the α family (CXC) primarily participate in chemotaxis functions, whereas those in the β family (CC) provide priming signal for immune cells. 76 For instance, CXCL10 is involved in T-lymphocyte recruitment after SCI, which contributes to post-traumatic tissue loss, 84 while CCL3 enhances the production of other pro-inflammatory cytokines through the G-protein coupled receptors CCR1, CCR4, and CCR5, leading to an exacerbation of inflammation that contributes to secondary tissue damage after SCI. 85 Taken together, the unregulated production of inflammatory mediators, albeit molecularly small, can lead to disastrous consequences toward functional recovery after SCI.

| Neurotrophic factors
The levels of growth promoting and inhibiting factors become disproportionate after SCI, resulting in an inhibitory environment within the spinal cord tissue. Neurotrophic molecules have been reported to enhance the survivability and proliferation capacity of neural cells and axonal regeneration within the spinal cord. 86 As such, an imbalance in these factors can lead to oligodendrocyte and neuronal death, as well as axonal degeneration. The most common neurotrophic factors include brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). 86,87 These neurotrophic mediators are synthesized as pro-peptides, which are cleaved intracellularly into mature neurotrophic proteins. 88 BDNF is a key molecule that plays a neuroprotective role by regulating synaptic plasticity and contributing to synaptic transmission. 89 However, its expression level is reduced drastically after SCI, and the overexpression of BDNF alleviates neuroinflammation through the induction of tyrosine kinase receptor B and phosphorylated p38. 90 NGF expression after SCI demonstrated improved behavioral outcomes by promoting axonal sprouting of the sensory afferents. 91 However, NGF has also been associated with neuropathic pain after nerve injury, where the binding of NGF to its receptors activates several downstream signaling pathways including the MAPK pathway. 92 This in turn led to the activation of NF-kB p65, which promotes the production of proinflammatory cytokines such as TNF-α and IL-1β, resulting in the development and maintenance of pain. 93,94 Interestingly, the pro-peptide of NGF, which is secreted in abundance after traumatic injuries, has been shown to reduce the number of oligodendrocytes through p75. 87,95 In addition, the complex formed between the precursor of NGF with Sortilin and p75 also triggers an apoptotic cascade. 96 Hence, the imbalance between neurotrophic factors and their precursors may also affect neural cell survival and death.

| Ionic imbalance
It is understood that biochemical events associated with secondary tissue damage include the disruptions of ionic homeostasis of K + , Na + , and Ca 2+ ion channels. 97 Following SCI, these channels are dysregulated due to damage to the cell membrane, as well as the release of proinflammatory mediators by immune cells. 98 Disrupting the myelin sheath of axons within the spinal cord tissue causes the imbalance of K + channels, which leads to further demyelination. 99 At the same time, the concentration of Na + becomes upregulated intracellularly, while K + and Mg 2+ become upregulated extracellularly, which eventually results in cellular edema. 100 This ionic imbalance further triggers intracellular phospholipase activity and acidosis. 101 Specifically, damaged neurons after SCI release high concentrations of glutamate neurotransmitter, causing Ca 2+ dysregulation, which compromises cellular machinery while increasing neural cell death. [102][103][104] Overall, ion imbalance plays a vital role in regulating the pathophysiology changes after SCI.

| MANIPULATING NEUROINFLAMMATION TO TREAT SCI
Extensive attempts have been made in modulating neuroinflammation to improve recovery after SCI, either through blockade of detrimental immune cell functions and neurotoxic pathways or enhancing the production of reparative and restorative cells and molecules. These approaches range from molecular-, cell-or biomaterial-based therapies that target different aspects of neuroinflammation after SCI.

| Depletion of immune cells and mediators
Therapeutic interventions that target specific cell types or intracellular signaling pathways have demonstrated positive prognosis in treating SCI. Neuroprotection can be achieved through the attenuation of peripheral immune cells infiltration by targeting adhesion molecules that are expressed on the surface of monocytes and/or neutrophils, which can rescue the capacity of donor cell populations to promote locomotor improvement after SCI. [105][106][107][108] For instance, antibodies that target CD11d/CD18 or α4β1 integrins expressed on monocyte, macrophages, and CD11d expressing microglia disrupt monocyteendothelial cell interactions and reduce both microglia and macrophage accumulation within the lesion site, leading to a reduction in tissue loss and increased functional recovery after SCI in rodent models. [109][110][111][112] The use of anti-Ly6G antibodies to deplete neutrophils has also led to improved recovery outcomes. 108 Depletion of both neutrophils and monocytes showed an early reduction in oxidative stress, nonheme iron, and expression of MMP-9 and stabilization of the BSCB, and thus greatly promoting neurological healing. 107 However, due to the double-edged nature of neuroinflammation, some studies have also shown a negative impact on wound healing and neurological outcomes when neutrophils are depleted. 113,114 Depletion of B-lymphocyte with therapeutic CD20 antibodies, such as rituximab or obinutuzumab, has also been used in modulating neuroinflammation and immunological events associated with SCI by reducing cell death and nitric oxide level. 115 These monoclonal antibodies also inhibit constitutive NF-kB signaling pathways by reducing the phosphorylation of components involved in the NF-kB pathway. 116 This is crucial as NF-kB is one of the pivotal mediators of pro-inflammatory gene expression, as well as the transcription of pro-inflammatory cytokines, chemokines, and adhesion molecules. 117 In addition, therapeutic CD20 antibodies also led to lower expressions of TNFα and IL-1β, which are associated with damage after SCI. 76,77,115 As B-lymphocytes have a role in trafficking T-cells into the CNS, 118 earlier findings have indicated that treatment with CD20 antibodies also affected T-lymphocyte activation, plausibly due to a decrease in antigen presentation by B-lymphocytes after depletion. 119 Meanwhile, directly depleting T-lymphocytes by split-dose gamma radiation after thymectomy in 4-week-old rats may also enhance neuronal survival after SCI. 120 Other than depleting immune cells or their adhesion factors, inhibition of cytokines or chemokines is another approach for limiting leukocyte infiltration and alleviating neuroinflammation. For instance, treatment with a broad-spectrum chemokine receptor antagonist, vMIP-II, reduces leukocyte influx and astrogliosis, while increasing axon and myelin sparing, and neuronal survival. 121,122 In addition, blocking the pro-inflammatory cytokine, IL-6, that promotes macrophages activation may also improve SCI recovery. Specifically, the monoclonal antibody, MR16-1, that targets IL-6 cytokine leads to the reduction of iNOS-and CD16/32+ macrophages, while promoting arginase-1-and CD206+ macrophages. 74 Interestingly, the effects of IL-6 inhibition are not only limited to macrophage or microglia, as it also alters astrocyte activation and ameliorates functional recovery after SCI. 75,123 Antagonizing CXCL10, the chemokine that is responsible for T-lymphocyte recruitment, has led to reduced neuronal death, an increase in axonal regeneration, and improve functional recovery after SCI. 121 Furthermore, anti-CXCL10 treatment also decreases the number of macrophages and B-lymphocytes. 124 The use of infliximab, which targets the pro-inflammatory cytokine, TNFα, as well as the genetic deletion of TNFα receptors drastically reduce neuroinflammation and oxidative injury while ameliorating neuropathic pain after SCI. 125,126 Exogenous administration of IL-1 receptor antagonist also led to a reduction in apoptosis and blocks p38 mitogen-activated protein kinase pathway. 127 Collectively, these findings suggest that targeting the inflammatory pathways is an alternative to improve neuroprotection and recovery after SCI.

| Promoting or transplanting cells with reparative and restorative functions
Another approach to improve functions after SCI focuses on immunomodulation and promotion of reparative immune cells such as the anti-inflammatory macrophages, either by pharmacological or transplantation therapies.
Pharmacological agents have been widely used to promote SCI recovery by reducing inflammation and redirecting immune cells toward the reparative pathway. One commonly used macrolide antibiotic, Azithromycin, has been reported to promote anti-inflammatory macrophage activation, which limits the secondary injury process after SCI, leading to improved tissue recovery. 128,129 Another antiinflammatory drug, minocycline, when administered acutely in a SCI rodent model has efficiently modulated the resident microglia to reduce its pro-inflammatory response while maintaining a proregenerative environment. 130 Exogeneous administration of Maresin 1, a highly conserved specialized pro-resolving mediator, has been demonstrated to resolve inflammatory responses by downregulating pro-inflammatory cytokines such as CXCL1, CXCL2, CCL3, CCL4, IL-6, and CSF3, silencing major inflammatory intracellular signaling pathways such as STAT1, STAT3, STAT5, p38, and ERK1/2, as well as altering macrophage activation toward the anti-inflammatory phenotype. 131 A more recent and comprehensive review on other immunomodulatory agents in spinal cord injury can be found in Wu et al. 132 Stem cell therapies have recently garnered attention for SCI treatment due to their capability to differentiate and replace degenerated neural cells. 133 Transplanted stem cells have been shown to promote neuro-and vascular-protective outcomes at different phases of SCI. 134 In addition to reorganizing the neuronal network, these cells also reduce local and systemic inflammation, support axonal regeneration and synaptic sprouting, and reduce glial scars. 134 The mechanisms of stem cell therapy are categorized into three distinct roles: (1) [158][159][160] The benefits of these calcineurin inhibitors are further enhanced when used in combination. 161,162 However, there remain several challenges in drug delivery to ameliorate neuroinflammation. For instance, the majority of the noninvasive route of drug delivery is less efficient in accessing the CNS, including the spinal cord, due to the presence of a BSCB. 163 In addition, most of the bioactive compounds that can pass through the BSCB are lipophilic, which may have reduced stability and half-lives under physiological conditions, resulting in difficulties to maintain an optimal dosage. 164 More importantly, drug diffusion within the host may lead to off-target effects, which has been reported with corticosteroids, where patients experienced severe side effects such as seizure, pneumonia, and haematemesis. 165 As clinical trials of corticosteroids in SCI have been relatively small, with an emphasis on subgroup effects, the use of corticosteroids in SCI should remain an area of controversy. 165 Thus, the involvement of biomaterial-based approaches may help overcome some of the challenges faced during drug delivery.

| Localized drug delivery
To tackle the challenges in drug delivery to the injured spinal cord, noninvasive strategies utilizing drug-loaded nanoparticles have been developed to overcome the BSCB. [166][167][168] In recent years, nanoparticles with neuroinflammation-targeting designs allowed more targeted delivery and had led to better recovery. 169,170 On the other hand, although it is more invasive, delivering the drugs in situ can bypass the BSCB and reach the injured site directly. Combined with a controlled-release mechanism, localized drug delivery can reduce the potential side effects of the immunomodulation drugs. For instance, loading anti-inflammation drugs in scaffolds or combining drug-loaded micro/nanoparticles with a hydrogel had demonstrated a reduction in microglia/macrophages activation and pro-inflammatory interleukins by ensuring that the local concentration of the drug is high enough to have a therapeutic effect (Table 2). 171,172,[175][176][177][178]180,187,189,194,196 More importantly, the particles can be designed to selectively target the microglia/ macrophages and control uptake kinetics by changing surface charge. 176,197 Other than low molecular weight anti-inflammatory drugs, scaffolds loaded with growth factors, microRNAs, and anti-inflammatory cytokine-encoding lentivirus also showed promising effects in reducing macrophage/microglial activation and improving functional recovery. 185,190,191 These growth factors and microRNAs also have a direct effect on stimulating nerve regeneration, which makes them ideal candidates that could have a synergistic effect in both anti-inflammation and nerve regeneration.

| Scaffolds for cell delivery
In addition to drug delivery, tissue engineering scaffolds have emerged as a powerful platform in combination with cell-based therapies as a form of regenerative intervention. A central component of tissue engineering is the use of biomaterials as a vehicle for cell transplantation by providing mechanical stability and support for cell adhesion and migration or recruiting endogenous progenitor cells from the surrounding tissues. 198 When the scaffolds are used to deliver cells, biomaterial scaffolds and cells synergistically controlled immune response and tissue regeneration (Table 3). 199,[203][204][205][207][208][209][210][211][212][213] Notably, mesenchymal stem cells secrete immunomodulating substances such as exosomes and CCL-2 to convert the macrophages/ microglia into anti-inflammatory phenotypes. 193,200,212 However, some implanted materials can evoke the host inflammatory response as they are regarded as foreign bodies that have been introduced to the site of lesion. 214 Hence, it would be highly beneficial to design the SCI scaffolds to be immunomodulatory through manipulating material chemistry and mechanical properties before combining with cells and drugs to achieve better recovery outcomes.

| Material chemistry
Traditionally, implantable biomaterials have been designed to be biocompatible by evading the immune system and minimizing foreign body responses. Earlier studies on implants in the CNS found that many of the materials and coatings might be pro-inflammatory and have low biocompatibility. 215 To improve material biocompatibility, low protein-binding coatings such as alginate could be useful in reducing microglial attachment. 215 However, such an approach also limits the attachment of other neural cells that are essential for regeneration. Consequently, the focus has shifted toward exploiting the properties of the biomaterials to modulate the immune response and immune cell phenotypes to achieve the desired outcomes such as better regeneration. 216 While anti-inflammatory effects were evaluated in most scaffolds in the form of reduced macrophage/microglial activation, more recent materials and scaffolds designed for SCI were increasingly assessing pro-and anti-inflammatory phenotypic switching as a feature of immunomodulation. Thus far, the majority of the natural materials used including decellularized extracellular matrices (ECM), collagen, laminin, chitosan, hyaluronic acid (HA), gelatin, and fibrin have well-documented biocompatibility and anti-inflammatory effects (Table 1). [230][231][232] Furthermore, some of these materials such as collagen, chitosan fragments, high molecular weight HA can reduce activation of macrophages, microglia, and astrocytes while polarizing macrophages toward the anti-inflammatory phenotypes. 209,217,221,233,234 Likewise, scaffolds developed based on decellularized tissue are rich with ECM proteins and hence can promote antiinflammatory macrophage polarization and recruit CD4+ Th2 Tlymphocytes to provide a pro-regenerative environment. 208,219,[235][236][237] This is particularly crucial for cell delivery where small molecules produced by activated T-lymphocytes might be cytotoxic to the grafted cells. 238 Synthetic materials such as polyurethane (PU), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), graphene oxide, and imidazole-polyorganophosphazenes, which have been used as scaffold materials for SCI regeneration, have also been assessed to reduce inflammation. 191,194,223,226,239 Although the antiinflammatory macrophages were observed in some of these scaffolds, the mechanism of how the materials polarize the macrophages is less clear. 223,226 Long-term evaluation is also needed to confirm that the products from polymers degradation do not elicit an additional inflammatory response. Furthermore, caution should be exercised regarding the hydrophilicity of the polymer surfaces as monocytes/macrophages adhere better onto hydrophobic surfaces. 240,241 Therefore, it is desired to use coatings or additives to better control the immune response towards the polymer surfaces. In particular, ECM proteins or ECM-derived peptides, which are effective in modulating macrophages, T lymphocytes, and B lymphocytes towards the anti- Note: The phenotypes of macrophages and microglia are presented as reported by the respective studies. In these studies, M1 typically refers to the proinflammatory phenotypes whereas M2 typically refers to the anti-inflammatory phenotypes.
T A B L E 3 Selected scaffolds for cell delivery with immunomodulation features after spinal cord injury

| Porosity and surface topography
Apart from having a tissue-compliant stiffness, for better integration with host tissue and to provide contact guidance, scaffolds are usually designed to allow efficient cell infiltration, in which pore size was also found to regulate macrophage phenotypes. [256][257][258][259] Otherwise, the scaffolds may elicit FBR, which in turn leads to larger glial scar or cyst formation. In addition to the macroarchitecture of the scaffolds, the microarchitecture of the scaffolds is also crucial in modulating the immune response through the surface topography of the implants. 231 The responses of neural cells toward surface topography are frequently exploited for neural tissue engineering but less consideration has been placed on the inflammatory response post-SCI. 260 Macrophage phenotype can be modulated by regulating cell shape through micro or nanopattern topographical cues. 261 Specifically, the elongated macrophages on the 400-500 nm wide nanopatterned grooves were driven toward an anti-inflammatory phenotype. 262 Similarly, electrospun nanofiber scaffold has served as an alternative to providing topographical stimuli. In particular, a reduced number of macrophages, macrophage activation, and secretion of pro-inflammatory molecules were found on PLA nanofiber (ø 600 nm) scaffolds as compared to films and microfibrous (ø 1.6 μm) scaffolds. 263 Similar results were also observed with PCL scaffolds. As compared to PCL films and random nanofibers, the aligned nanofibers (ø 506 nm) scaffolds had reduced monocyte/macrophage adhesion and a thinner fibrous capsule in vivo. 264 Recently, in a transplanted nanofiber-hydrogel composite scaffold for SCI treatment, anti-inflammatory macrophages were found to be present predominantly in the areas with the nanofibers, suggesting the possible role of nanofibers directly modulating immune cells phenotype. 228 On the other hand, while less is known about regulating lymphocytes and neutrophils through surface topography, lymphocytes and neutrophils found on implants with rough surfaces, created through sandblasting followed by acid-etching or physical scratching, secreted less pro-inflammatory cytokines. [265][266][267] In particular, rough and hydrophilic surfaces polarized the adaptive immune system toward the pro-regenerative Th2 phenotype mediated by macrophages. 267 Similar to macrophages, nanofiber topography has a positive effect on astrocytes as nanofiber topography promoted astrocyte adhesion with downregulated GFAP expression, leading to reduced F I G U R E 2 Biomaterial-based therapies to modulate neuroinflammation and treat SCI. The combination of biomaterial design, drug delivery, cell therapy, and rehabilitation can be utilized to target neuroinflammation and neuroregeneration to achieve a synergistic effect in promoting functional recovery after SCI. Illustrations are created with BioRender.com. astrocytes activity. 239 Aligned electrospun fiber topography (ø 2.4 μm) also directed astrocytic migration and increased the rates of glutamate uptake as a readout for neuroprotective effect. 268  populations, scaffolds with a sequential delivery mechanism of drugs or physical signals targeting different stages could be more effective in promoting nerve regeneration and motor recovery after SCI. 194,288 Current immunomodulation approaches for treating SCI are mainly through immune response reduction and macrophage phenotypic shift. [289][290][291][292] It will be valuable to assess other immune cells and responses as well as target these mediators for better nerve regeneration. As discussed earlier, future scaffold designs may benefit from referring to the biomaterial approaches in targeting autoimmune diseases, graft rejection, and inflammation in other tissues. 216,282,[293][294][295][296][297] Finally, including a rehabilitation regimen would also be beneficial as rehabilitation and scaffold implantation was found to synergistically promote the skewing of macrophage phenotype toward antiinflammatory phenotypes and better functional recovery. 298 NRF2019-THE002-0001).

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.